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Mechanical loading prevents the stimulating effect of IL-1β on osteocyte-modulated osteoclastogenesis
Biochemical and Biophysical Research Communications 420 (2012) 11–16
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mechanical loading prevents the stimulating effect of IL-1b on osteocyte-modulated osteoclastogenesis Rishikesh N. Kulkarni, Astrid D. Bakker, Vincent Everts, Jenneke Klein-Nulend ⇑ Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history: Received 31 January 2012 Available online 27 February 2012 Keywords: Interleukin-1b Osteocytes Osteoclastogenesis Mechanical loading Pulsating fluid flow
a b s t r a c t Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1b (IL-1b), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1b affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1b (0.1–1 ng/ml) for 24 h. Cells were either or not subjected to mechanical loading by 1 h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) in the presence of IL-1b (0.1–1 ng/ml). Conditioned medium was collected after 1 h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1b-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1b-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/ CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1b, as well as conditioned medium from static IL-1b-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL-1btreated osteocytes prevented osteoclast formation. Incubation with IL-1b upregulated RANKL and downregulated OPG gene expression by static osteocytes. PFF upregulated CYR61, RANKL, and OPG gene expression by osteocytes. Our results suggest that IL-1b increases osteocyte-modulated osteoclastogenesis, and that mechanical loading of osteocytes may abolish IL-1b-induced osteoclastogenesis. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Bones are subjected to a variety of mechanical loads during daily activities. Bone mass and architecture are continuously adapted to the daily mechanical loads. Osteocytes play an important role in the adaptation of bone to mechanical loading, by sensing the mechanical loads and orchestrating the activity of bone-forming osteoblasts and bone-resorbing osteoclasts [1–3]. Osteocytes are thought to regulate bone mass by orchestrating the balance between bone formation and resorption in response to mechanical loading [4]. Thus, any factor that alters the response of osteocytes to mechanical loading potentially affects bone mass. Inflammatory diseases such as rheumatoid arthritis (RA) are often accompanied by higher plasma and synovial fluid levels of
⇑ Corresponding author at: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 Amsterdam, LA, The Netherlands. E-mail address: [email protected] (J. Klein-Nulend). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.099
interleukin-1b (IL-1b) [5,6], and increased osteoclastic bone resorption [7]. Bone loss in patients with RA might be caused by the physiologic adaptation of bone to reduced physical activity, and/or by the use of corticosteroids by these patients. It could also be a direct effect of increased IL-1b levels. The relative importance of the proinflammatory cytokines IL-1 and TNF in the pathogenesis of RA is the subject of much debate. TNF is more important in inflammatory processes whereas IL-1 plays a more important role in joint destruction in a murine arthritis model [8]. The proinflammatory cytokine IL-1b facilitates osteoclastogenesis by inducing expression of receptor activator of nuclear factor kappa B (RANKL) by osteoblasts [9]. RANKL stimulates osteoclast precursors to commit to the osteoclastic phenotype whereas osteoprotegerin (OPG) inhibits RANKL activity [10]. Crockett et al. have shown that cysteine rich protein 61 (CYR61/CCN1) inhibits the formation of osteoclasts [11]. CYR61 is an extracellular matrix-associated signaling protein that belongs to the connective tissue growth factor (CCN) family of proteins. CYR61 stimulates osteoblast proliferation and
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differentiation and is upregulated during fracture repair [12,13]. It is yet unknown whether IL-1b modulates CYR61 expression. Several studies have shown that nitric oxide production by bone cells is rapidly increased in response to mechanical stress in vitro [14,15]. Nitric oxide mediates adaptive bone formation [16], and is essential for the inhibition of osteoclastogenesis by mechanically loaded MLO-Y4 osteocytes [17]. Bakker et al. have shown that both TNFa and IL-1b inhibit the upregulation of nitric oxide production after mechanical stimulation by PFF [18]. Therefore IL-1b might interfere with adaptive bone formation during inflammatory diseases by affecting osteocyte-modulated osteoclastogenesis. However, whether mechanical loading of osteocytes in the presence of IL-1b affects osteocyte-modulated osteoclastogenesis is unknown. Since IL-1b alters the mechano-response of osteocytes we aimed to investigate whether IL-1b affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. 2. Material and methods 2.1. Cell culture
or static culture. After 1 h PFF the cells were lysed for total RNA isolation as described below. 2.3. Osteoclast formation MLO-Y4 osteocytes were seeded at a density of 1 103 cells/ well in 48-well tissue culture plates (Greiner Bio-One, Frickenhausen, Germany). After attachment the osteocytes were either or not pre-incubated for 24 h with 0.1, 0.5, or 1 ng/ml of mouse recombinant IL-1b. After this 24 h period, the medium containing IL-1b was removed, and 1 106 bone marrow cells/cm2 were seeded on top of the IL-1b treated osteocytes. The cells were kept in co-culture in a-MEM (Gibco) supplemented with 10% FBS and antibiotics. In another co-culture set-up, conditioned medium from mechanically loaded or static IL-1b treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Culture medium and osteocyte-conditioned medium were refreshed after 3 days. After 7 days of co-culture, cells were fixed in 4% formaldehyde in PBS for 10 min. Fixed cells were washed with PBS, and stained for tartrate-resistant acid phosphatase (TRACP) according to the manufacturer’s instructions (Sigma). The number of TRACP-positive multinucleated (3 or more nuclei per cell) and mononuclear cells were counted using a Leica DM IL microscope (Leica, Wetzlar, Germany) equipped with a 20 objective.
MLO-Y4 osteocytes (kindly provided by Dr. L. Bonewald, San Antonio, TX, USA) [19] were cultured up to near-confluency in 75cm2 culture flasks using a-MEM supplemented with 5% fetal bovine serum (FBS; Gibco, Grand Island, NY), 5% calf serum (Gibco), 1.25 lg/ml fungizone (Gibco), 150 lg/ml penicillin (Sigma, St. Louis, MO), and 125 lg/ml streptomycin (Sigma) at 37 °C and 5% CO2 in air. Mouse bone marrow cells were used for the osteoclast formation assay. The animal committee of the VU University Amsterdam approved the use of mice in these experiments. Femurs and tibiae were harvested and soft tissue was removed as previously described in detail [20]. Cleaned femurs and tibiae were then ground in a mortar with a-MEM (Gibco) supplemented with 10% FBS, 150 lg/ml penicillin, 125 lg/ml streptomycin, 1.25 lg/ml fungizone, and heparin (170 IE/ml; Leo Pharmaceutical Products B.V., Weesp, The Netherlands). The resulting cell suspension was aspirated through a 21-gauge needle and filtered using a 100-lm pore size Cell Strainer filter (Falcon/Becton Dickinson, Franklin Lakes, NJ). Cells were then washed twice in culture medium, centrifuged for 10 min at 200g, and 1 106 cells/cm2 were seeded on top of either or not IL-1b-treated (0.1, 0.5, or 1 ng/ml) MLO-Y4 osteocytes.
Real-time polymerase chain reaction (PCR) was used to determine gene expression of CYR61, RANKL, OPG, and the housekeeping gene GAPDH (all primers from Applied Biosystems, Foster, CA). Total RNA was isolated using lysis buffer RA I (Macherey–Nagel, Düren, Germany) according to the manufacturer’s instructions. cDNA synthesis was performed using 0.5–1 lg of total RNA in a 20 ll reaction mixture consisting of 5 units of Transcriptor Reverse Transcriptase (Roche Diagnostics, Mannheim, Germany), 0.08 A260 units of random primers (Roche Diagnostics), 1 mM of each dNTP (InVitrogen, Calrsbad, CA), and 1x concentrated Transcriptor RT reaction buffer (Roche Diagnostics). Real time PCR reactions were performed using Taq-ManÒ Gene Expression assays (TaqManÒ, Applied Biosystems) in an ABI Prism 7700 DNA sequence detector (Applied Biosystems). Gene expression values were normalized for the housekeeping gene GAPDH.
2.2. Pulsatile fluid flow (PFF)
2.5. Statistics
MLO-Y4 osteocytes at passage 30 or 31 were harvested using 0.25% trypsin (Difco Laboratories, Detroit, MI) and 0.1% EDTA (Sigma) in PBS, and seeded at 2 104 cells/cm2 on polylysinecoated (50 lg/ml; poly-L-lysine hydrobromide; Sigma) glass slides (15 cm2) in a-MEM with 5% FBS, 5% calf serum, and antibiotics. Osteocytes were incubated with mouse recombinant IL-1b (0.1, 0.5, or 1 ng/ml) (Sigma) for 24 h before the start of the experiment. They were incubated overnight at 37 °C with 5% CO2 in air, and subjected to mechanical loading for 1 h by PFF or kept under static control conditions in the presence or absence of IL-1b. PFF at 5 Hz pulse frequency was generated by pumping 13 ml of culture medium in a pulsatile manner through a parallel-plate flow chamber (65 24 0.3 mm) containing the bone cells [21]. The mean applied fluid shear stress was 0.7 Pa with a pulse amplitude of 0.3 Pa, and the estimated peak stress rate was 8.4 Pa/s [22,23]. Control cultures were kept under stationary conditions in a petridish containing 13 ml flow medium under similar conditions as the experimental cultures, i.e. at 37 °C in a humidified atmosphere of 5% CO2 in air. Conditioned medium was collected after 1 h of PFF
One way-ANOVA was performed to assess whether there was a significant difference in mRNA expression and osteoclast formation between groups. Bonferroni comparison between pairs of groups was used as a post hoc test. Two way-ANOVA with pairwise comparison was used to study the effect of IL-1b and PFF on mRNA expression and osteoclast formation. Differences were considered significant when p < 0.05.
2.4. Analysis of gene expression
3. Results 3.1. IL-1b increases osteocyte-modulated osteoclastogenesis The effect of IL-1b on osteocyte-modulated osteoclastogenesis in co-cultures of IL-1b (0.1–1 ng/ml) treated osteocytes and mouse bone marrow cells was investigated. We found that IL-1b dosedependently increased the number of osteoclasts, as assessed by counting the number of TRACP-positive mononuclear cells after 7 days of co-culture (Fig. 1).
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We found that the conditioned medium from IL-1b-treated static osteocytes dose-dependently increased the number of osteoclasts (Fig. 3A). Conditioned medium from mechanically loaded osteocytes strongly inhibited the formation of osteoclasts (Fig. 3A and B). This inhibiting effect was seen in the absence and presence of different concentrations of IL-1b (Fig. 3A and B).
3.4. CYR61 is upregulated in mechanically stimulated osteocytes
Fig. 1. Effect of IL-1b on osteocyte-modulated osteoclastogenesis. IL-1b at 0.1, 0.5, and 1 ng/ml upregulated the number of TRACP-positive multinucleated cells after 7 days of co-culture of MLO-Y4 osteocytes and mouse bone marrow cells. Values are mean ± SEM from six separate experiments. CTL, control. ⁄Significant effect of IL-1b, p < 0.05.
3.2. IL-1b alters osteoclastogenesis-related gene expression by osteocytes Since IL-1b increased the formation of osteoclasts, we investigated whether IL-1b alters osteoclastogenesis-related gene expression by osteocytes. IL-1b (0.5 and 1 ng/ml) upregulated RANKL gene expression by 3.7- to 4.0-fold (Fig. 2A). However, IL-1b (0.5 and 1 ng/ml) downregulated OPG gene expression by 1.2- to 1.7fold (Fig. 2B). As a result IL-1b (0.5 and 1 ng/ml) upregulated RANKL/OPG ratio by 2.3- to 4.0-fold. IL-1b did not affect CYR61 gene expression (Fig. 2C). 3.3. Mechanical loading of osteocytes inhibits IL-1b stimulated osteoclastogenesis Next, we determined whether mechanical loading of osteocytes in the absence or presence of IL-1b affects osteoclast formation in a co-culture system of MLO-Y4 osteocytes and bone marrow cells.
To assess whether mechanical loading of osteocytes in the presence or absence of IL-1b affects CYR61, RANKL, and OPG gene expression, MLO-Y4 osteocytes were treated with or without 1 h PFF in the presence of IL-1b (0.1–1 ng/ml). PFF upregulated CYR61 gene expression by 1.3-fold in the absence of IL-1b (Fig. 4A). Addition of IL-1b (0.1–1 ng/ml) inhibited the upregulation of CYR61 gene expression after mechanical stimulation by PFF (Fig. 4A). PFF did not affect CYR61 gene expression in the presence of IL-1b (0.1–1 ng/ml) (Fig. 4A). PFF upregulated RANKL gene expression by 3.0-fold (Fig. 4B) and OPG gene expression by 1.4fold in the absence of IL-1b (Fig. 4C). Addition of IL-1b (0.5 and 1 ng/ml) inhibited the upregulation of OPG gene expression after mechanical stimulation by PFF (Fig. 4C). PFF upregulated RANKL gene expression by 1.6-fold in the presence of IL-1b (0.1 ng/ml), but did not affect OPG gene expression in the presence of IL-1b (0.1–1 ng/ml).
4. Discussion In the present study we found that IL-1b increases osteocytemodulated osteoclastogenesis. These results are in accordance with data by others showing that IL-1b indirectly facilitates osteoclastogenesis by acting on osteoblasts or human periodontal ligament fibroblasts [24,25]. We found that IL-1b upregulates RANKL and inhibits OPG gene expression by osteocytes. Our results show that IL-1b probably stimulates osteoclastogenesis by modulating RANKL and OPG gene expression by osteocytes. Alternatively the
Fig. 2. Effect of IL-1b on RANKL, OPG, and CYR61 gene expression by MLO-Y4 osteocytes. Cells were incubated with IL-1b (0.1–1 ng/ml) for 24 h. (A) IL-1 b at 0.5 and 1 ng/ml upregulated RANKL gene expression. (B) IL-1 b at 0.5 and 1 ng/ml downregulated OPG gene expression. (C) IL-1 b did not affect CYR61 gene expression. Values are mean ± SEM of IL-1b treated-over-control ratios from five different experiments. Dashed line, T/C = 1 (no effect of IL-1b). ⁄Significant effect of IL-1b, p < 0.05.
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Fig. 3. Effect of conditioned medium obtained from static or mechanically stimulated osteocytes treated with or without IL-1b on osteocyte-modulated osteoclastogenesis. Cells were treated with IL-1b for 24 h before application of PFF. (A) Conditioned medium from static osteocytes treated with 0.1, 0.5, or 1 ng/ml IL-1b significantly upregulated the number of TRACP-positive multinucleated cells, whereas conditioned medium from mechanically stimulated osteocytes treated with 0.1, 0.5, or 1 ng/ml IL-1b significantly inhibited the number of TRACP-positive multinucleated cells. (B) Conditioned medium from mechanically stimulated osteocytes inhibited the formation of osteoclasts. Values are mean ± SEM from four separate experiments. PFF, pulsating fluid flow; static, static control conditions; CTL, control. ⁄Significant effect of PFF, p < 0.05. # Significant effect of IL1-b, p < 0.05.
increased formation of osteoclast-like cells might have been due to osteocyte apoptosis [26]. Osteocytes extend their cell processes into bone marrow spaces [27], where osteoclast precursors are present. It is likely that osteocytes communicate with these osteoclast precursors present in the bone marrow space via their dendritic processes. Therefore we used a co-culture model of MLO-Y4 osteocytes and mouse bone marrow cells, which likely mimics the in vivo situation. Soluble factors released by mechanically loaded osteocytes inhibit osteoclastogenesis induced by stromal cells and osteocytes [28,29]. Therefore we investigated if conditioned medium from mechanically loaded and IL-1b-treated osteocytes does modulate osteoclastogenesis. We found that conditioned medium from static IL-1b-treated osteocytes dose-dependently increased the number of osteoclasts in co-cultures of osteocytes and bone marrow cells. Conversely conditioned medium from mechanically loaded IL-1b-treated osteocytes inhibited the formation of osteoclasts. To our knowledge we show for the first time that mechanical loading of osteocytes in the presence of IL-1b abolishes osteoclastogenesis. Since osteocytes are the most abundant bone cell type, vastly outnumbering the osteoblasts and osteoclasts in the bone, our findings provide important information related to the prevention of IL-1b-induced bone loss. Mechanically loaded osteocytes produce several factors which inhibit osteoclastogenesis. Osteocytes produce high levels of nitric oxide in response to mechanical stimulation [15,30], and nitric oxide mediates the inhibition of osteoclast activity by mechanically stimulated osteocytes [17]. Although IL-1b inhibits the upregulation of nitric oxide production after mechanical stimulation by PFF [18], we found that mechanical loading of osteocytes in the
presence of IL-1b inhibits osteoclastogenesis. We propose that the inhibition of IL-b-induced osteoclastogenesis by mechanically loaded osteocytes is independent of nitric oxide. Since a decrease in the RANKL/OPG ratio inhibits osteoclastogenesis, we also investigated whether mechanical loading of osteocytes in the presence of IL-1b modulates the RANKL/OPG ratio in osteocytes. We found that mechanical stimulation in the presence of IL-1b did not decrease the RANKL/OPG ratio in osteocytes. This suggests that mechanical loading of osteocytes in the presence of IL-1b might inhibit osteoclastogenesis in a RANKL/RANK/OPG independent manner. We then determined the effect of mechanical loading on CRY61 gene expression, a negative regulator of osteoclastogenesis [11], in osteocytes. Under these conditions we found an upregulation of CYR61 gene expression, which has not been shown before. Since CYR61 has been shown to inhibit osteoclastogenesis in a RANKL/RANK/OPG independent manner [11], this suggests that CYR61 might be a soluble factor produced after mechanical loading of osteocytes in the absence of IL-1b leading to the inhibition of osteoclastogenesis. However we found that IL-1b inhibits the upregulation of CYR61 gene expression after PFF. In spite of the inhibited expression of CYR61, conditioned medium from mechanically loaded IL-1b treated osteocytes still inhibited the formation of osteoclasts. Therefore CYR61 might not be involved in the inhibition of IL-1b-induced osteoclastogenesis. We also found that conditioned medium from mechanically loaded IL-1b-treated osteocytes inhibited clustering of osteoclast precursors (data not shown). This finding suggests the presence of factors in the conditioned medium that inhibit migration of osteoclast precursors towards each other to form osteoclasts.
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Fig. 4. Effect of 1 h PFF on CYR61, RANKL, and OPG gene expression by MLO-Y4 osteocytes in the presence or absence of IL-1b. (A) PFF upregulated CYR61 gene expression by osteocytes in the absence of IL-1b. (B) PFF upregulated RANKL gene expression by osteocytes in the absence and presence of 0.5 ng/ml IL-1b. (C) PFF upregulated OPG gene expression by osteocytes in the absence of IL-1b. Values are mean ± SEM from five different PFF experiments. PFF, pulsating fluid flow; static, static control conditions; CTL, control. ⁄Significant effect of PFF, p < 0.05. #Significant effect of IL1-b, p < 0.05.
Further research focusing on the identification of the specific soluble factors produced after mechanical loading of IL-1b-treated osteocytes that inhibit osteoclastogenesis is warranted. In conclusion, our results show that IL-1b increases osteocytemodulated osteoclastogenesis, whereas mechanical loading of osteocytes inhibits IL-1b-induced osteoclastogenesis. Based on these findings we propose that in patients with RA, limited movement or disuse as a result of pain and presence of IL-1b might enhance bone loss, whereas mechanical loading of bone in these patients will hopefully, at least in part, alleviate the negative effects of IL-1b on bone mass. Acknowledgments The work of R.N. Kulkarni was supported by a Grant from the University of Amsterdam. The Research Institute MOVE of the VU University Amsterdam supported the work of A.D. Bakker. The authors thank J.M.A. Hogervorst for excellent help in performing mechanical loading experiments. References [1] T. Nakashima, M. Hayashi, T. Fukunaga, et al., Evidence for osteocyte regulation of bone homeostasis through RANKL expression, Nat. Med. 17 (2011) 1231– 1234. [2] P.S. Vezeridis, C.M. Semeins, Q. Chen, J. Klein-Nulend, Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation, Biochem. Biophys. Res. Commun. 348 (2006) 1082–1088. [3] J. Xiong, M. Onal, R.L. Jilka, et al., Matrix-embedded cells control osteoclast formation, Nat. Med. 17 (2011) 1235–1241. [4] E.H. Burger, J. Klein-Nulend, Mechanotransduction in bone – role of the lacunocanalicular network, FASEB J. 13 (1999) S101–S112. [5] J.A. Eastgate, J.A. Symons, N.C. Wood, et al., Correlation of plasma interleukin 1 levels with disease activity in rheumatoid arthritis, Lancet 24 (1988) 706–709.
[6] P. Kahle, J.G. Saal, K. Schaudt, et al., Determination of cytokines in synovial fluids: correlation with diagnosis and histomorphological characteristics of synovial tissue, Ann. Rheum. Dis. 51 (1992) 731–734. [7] M. Bromley, D.E. Woolley, Chondroclasts and osteoclasts at subchondral sites of erosion in the rheumatoid joint, Arthritis Rheum. 27 (1984) 968–975. [8] L.A. Joosten, M.M. Helsen, T. Saxne, et al., IL-1 alpha beta blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-alpha blockade only ameliorates joint inflammation, J. Immunol. 163 (1999) 5049–5055. [9] S. Jurado, N. Garcia-Giralt, A. Díez-Pérez, et al., Effect of IL-1beta, PGE(2), and TGF-beta1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts, J. Cell. Biochem. 15 (2010) 304–310. [10] S. Teitelbaum, Osteoclasts: what do they do and how do they do it?, Am J. Pathol. 170 (2007) 427–435. [11] J.C. Crockett, N. Schütze, D. Tosh, et al., The matricellular protein CYR61 inhibits osteoclastogenesis by a mechanism independent of alphavbeta3 and alphavbeta5, Endocrinology 148 (2007) 5761–5768. [12] W. Si, Q. Kang, H.H. Luu, et al., CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells, Mol. Cell. Biol. 26 (2006) 2955– 2964. [13] J. Lienau, H. Schell, D.R. Epari, et al., CYR61 (CCN1) protein expression during fracture healing in an ovine tibial model and its relation to the mechanical fixation stability, J. Orthop. Res. 24 (2006) 254–262. [14] A.A. Pitsillides, S.C. Rawlinson, R.F. Suswillo, et al., Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?, FASEB J 9 (1995) 1614–1622. [15] J. Klein-Nulend, C.M. Semeins, N.E. Ajubi, et al., Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblastscorrelation with prostaglandin upregulation, Biochem. Biophys. Res. Commun. 217 (1995) 640–648. [16] S.W. Fox, T.J. Chambers, J.W. Chow, Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation, Am. J. Physiol. 270 (1996) E955–E960. [17] S.D. Tan, T.J. de Vries, A.M. Kuijpers-Jagtman, et al., Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption, Bone 41 (2007) 745–751. [18] A.D. Bakker, V.C. Silva, R. Krishnan, et al., Tumor necrosis factor alpha and interleukin-1beta modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes, Arthritis Rheum. 60 (2009) 3336–3345. [19] Y. Kato, J.J. Windle, B.A. Koop, et al., Establishment of an osteocyte-like cell line, MLO-Y4, J. Bone Miner. Res. 12 (1997) 2014–2023.
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Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mechanical loading prevents the stimulating effect of IL-1b on osteocyte-modulated osteoclastogenesis Rishikesh N. Kulkarni, Astrid D. Bakker, Vincent Everts, Jenneke Klein-Nulend ⇑ Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history: Received 31 January 2012 Available online 27 February 2012 Keywords: Interleukin-1b Osteocytes Osteoclastogenesis Mechanical loading Pulsating fluid flow
a b s t r a c t Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1b (IL-1b), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1b affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1b (0.1–1 ng/ml) for 24 h. Cells were either or not subjected to mechanical loading by 1 h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) in the presence of IL-1b (0.1–1 ng/ml). Conditioned medium was collected after 1 h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1b-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1b-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/ CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1b, as well as conditioned medium from static IL-1b-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL-1btreated osteocytes prevented osteoclast formation. Incubation with IL-1b upregulated RANKL and downregulated OPG gene expression by static osteocytes. PFF upregulated CYR61, RANKL, and OPG gene expression by osteocytes. Our results suggest that IL-1b increases osteocyte-modulated osteoclastogenesis, and that mechanical loading of osteocytes may abolish IL-1b-induced osteoclastogenesis. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Bones are subjected to a variety of mechanical loads during daily activities. Bone mass and architecture are continuously adapted to the daily mechanical loads. Osteocytes play an important role in the adaptation of bone to mechanical loading, by sensing the mechanical loads and orchestrating the activity of bone-forming osteoblasts and bone-resorbing osteoclasts [1–3]. Osteocytes are thought to regulate bone mass by orchestrating the balance between bone formation and resorption in response to mechanical loading [4]. Thus, any factor that alters the response of osteocytes to mechanical loading potentially affects bone mass. Inflammatory diseases such as rheumatoid arthritis (RA) are often accompanied by higher plasma and synovial fluid levels of
⇑ Corresponding author at: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 Amsterdam, LA, The Netherlands. E-mail address: [email protected] (J. Klein-Nulend). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.099
interleukin-1b (IL-1b) [5,6], and increased osteoclastic bone resorption [7]. Bone loss in patients with RA might be caused by the physiologic adaptation of bone to reduced physical activity, and/or by the use of corticosteroids by these patients. It could also be a direct effect of increased IL-1b levels. The relative importance of the proinflammatory cytokines IL-1 and TNF in the pathogenesis of RA is the subject of much debate. TNF is more important in inflammatory processes whereas IL-1 plays a more important role in joint destruction in a murine arthritis model [8]. The proinflammatory cytokine IL-1b facilitates osteoclastogenesis by inducing expression of receptor activator of nuclear factor kappa B (RANKL) by osteoblasts [9]. RANKL stimulates osteoclast precursors to commit to the osteoclastic phenotype whereas osteoprotegerin (OPG) inhibits RANKL activity [10]. Crockett et al. have shown that cysteine rich protein 61 (CYR61/CCN1) inhibits the formation of osteoclasts [11]. CYR61 is an extracellular matrix-associated signaling protein that belongs to the connective tissue growth factor (CCN) family of proteins. CYR61 stimulates osteoblast proliferation and
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differentiation and is upregulated during fracture repair [12,13]. It is yet unknown whether IL-1b modulates CYR61 expression. Several studies have shown that nitric oxide production by bone cells is rapidly increased in response to mechanical stress in vitro [14,15]. Nitric oxide mediates adaptive bone formation [16], and is essential for the inhibition of osteoclastogenesis by mechanically loaded MLO-Y4 osteocytes [17]. Bakker et al. have shown that both TNFa and IL-1b inhibit the upregulation of nitric oxide production after mechanical stimulation by PFF [18]. Therefore IL-1b might interfere with adaptive bone formation during inflammatory diseases by affecting osteocyte-modulated osteoclastogenesis. However, whether mechanical loading of osteocytes in the presence of IL-1b affects osteocyte-modulated osteoclastogenesis is unknown. Since IL-1b alters the mechano-response of osteocytes we aimed to investigate whether IL-1b affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. 2. Material and methods 2.1. Cell culture
or static culture. After 1 h PFF the cells were lysed for total RNA isolation as described below. 2.3. Osteoclast formation MLO-Y4 osteocytes were seeded at a density of 1 103 cells/ well in 48-well tissue culture plates (Greiner Bio-One, Frickenhausen, Germany). After attachment the osteocytes were either or not pre-incubated for 24 h with 0.1, 0.5, or 1 ng/ml of mouse recombinant IL-1b. After this 24 h period, the medium containing IL-1b was removed, and 1 106 bone marrow cells/cm2 were seeded on top of the IL-1b treated osteocytes. The cells were kept in co-culture in a-MEM (Gibco) supplemented with 10% FBS and antibiotics. In another co-culture set-up, conditioned medium from mechanically loaded or static IL-1b treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Culture medium and osteocyte-conditioned medium were refreshed after 3 days. After 7 days of co-culture, cells were fixed in 4% formaldehyde in PBS for 10 min. Fixed cells were washed with PBS, and stained for tartrate-resistant acid phosphatase (TRACP) according to the manufacturer’s instructions (Sigma). The number of TRACP-positive multinucleated (3 or more nuclei per cell) and mononuclear cells were counted using a Leica DM IL microscope (Leica, Wetzlar, Germany) equipped with a 20 objective.
MLO-Y4 osteocytes (kindly provided by Dr. L. Bonewald, San Antonio, TX, USA) [19] were cultured up to near-confluency in 75cm2 culture flasks using a-MEM supplemented with 5% fetal bovine serum (FBS; Gibco, Grand Island, NY), 5% calf serum (Gibco), 1.25 lg/ml fungizone (Gibco), 150 lg/ml penicillin (Sigma, St. Louis, MO), and 125 lg/ml streptomycin (Sigma) at 37 °C and 5% CO2 in air. Mouse bone marrow cells were used for the osteoclast formation assay. The animal committee of the VU University Amsterdam approved the use of mice in these experiments. Femurs and tibiae were harvested and soft tissue was removed as previously described in detail [20]. Cleaned femurs and tibiae were then ground in a mortar with a-MEM (Gibco) supplemented with 10% FBS, 150 lg/ml penicillin, 125 lg/ml streptomycin, 1.25 lg/ml fungizone, and heparin (170 IE/ml; Leo Pharmaceutical Products B.V., Weesp, The Netherlands). The resulting cell suspension was aspirated through a 21-gauge needle and filtered using a 100-lm pore size Cell Strainer filter (Falcon/Becton Dickinson, Franklin Lakes, NJ). Cells were then washed twice in culture medium, centrifuged for 10 min at 200g, and 1 106 cells/cm2 were seeded on top of either or not IL-1b-treated (0.1, 0.5, or 1 ng/ml) MLO-Y4 osteocytes.
Real-time polymerase chain reaction (PCR) was used to determine gene expression of CYR61, RANKL, OPG, and the housekeeping gene GAPDH (all primers from Applied Biosystems, Foster, CA). Total RNA was isolated using lysis buffer RA I (Macherey–Nagel, Düren, Germany) according to the manufacturer’s instructions. cDNA synthesis was performed using 0.5–1 lg of total RNA in a 20 ll reaction mixture consisting of 5 units of Transcriptor Reverse Transcriptase (Roche Diagnostics, Mannheim, Germany), 0.08 A260 units of random primers (Roche Diagnostics), 1 mM of each dNTP (InVitrogen, Calrsbad, CA), and 1x concentrated Transcriptor RT reaction buffer (Roche Diagnostics). Real time PCR reactions were performed using Taq-ManÒ Gene Expression assays (TaqManÒ, Applied Biosystems) in an ABI Prism 7700 DNA sequence detector (Applied Biosystems). Gene expression values were normalized for the housekeeping gene GAPDH.
2.2. Pulsatile fluid flow (PFF)
2.5. Statistics
MLO-Y4 osteocytes at passage 30 or 31 were harvested using 0.25% trypsin (Difco Laboratories, Detroit, MI) and 0.1% EDTA (Sigma) in PBS, and seeded at 2 104 cells/cm2 on polylysinecoated (50 lg/ml; poly-L-lysine hydrobromide; Sigma) glass slides (15 cm2) in a-MEM with 5% FBS, 5% calf serum, and antibiotics. Osteocytes were incubated with mouse recombinant IL-1b (0.1, 0.5, or 1 ng/ml) (Sigma) for 24 h before the start of the experiment. They were incubated overnight at 37 °C with 5% CO2 in air, and subjected to mechanical loading for 1 h by PFF or kept under static control conditions in the presence or absence of IL-1b. PFF at 5 Hz pulse frequency was generated by pumping 13 ml of culture medium in a pulsatile manner through a parallel-plate flow chamber (65 24 0.3 mm) containing the bone cells [21]. The mean applied fluid shear stress was 0.7 Pa with a pulse amplitude of 0.3 Pa, and the estimated peak stress rate was 8.4 Pa/s [22,23]. Control cultures were kept under stationary conditions in a petridish containing 13 ml flow medium under similar conditions as the experimental cultures, i.e. at 37 °C in a humidified atmosphere of 5% CO2 in air. Conditioned medium was collected after 1 h of PFF
One way-ANOVA was performed to assess whether there was a significant difference in mRNA expression and osteoclast formation between groups. Bonferroni comparison between pairs of groups was used as a post hoc test. Two way-ANOVA with pairwise comparison was used to study the effect of IL-1b and PFF on mRNA expression and osteoclast formation. Differences were considered significant when p < 0.05.
2.4. Analysis of gene expression
3. Results 3.1. IL-1b increases osteocyte-modulated osteoclastogenesis The effect of IL-1b on osteocyte-modulated osteoclastogenesis in co-cultures of IL-1b (0.1–1 ng/ml) treated osteocytes and mouse bone marrow cells was investigated. We found that IL-1b dosedependently increased the number of osteoclasts, as assessed by counting the number of TRACP-positive mononuclear cells after 7 days of co-culture (Fig. 1).
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We found that the conditioned medium from IL-1b-treated static osteocytes dose-dependently increased the number of osteoclasts (Fig. 3A). Conditioned medium from mechanically loaded osteocytes strongly inhibited the formation of osteoclasts (Fig. 3A and B). This inhibiting effect was seen in the absence and presence of different concentrations of IL-1b (Fig. 3A and B).
3.4. CYR61 is upregulated in mechanically stimulated osteocytes
Fig. 1. Effect of IL-1b on osteocyte-modulated osteoclastogenesis. IL-1b at 0.1, 0.5, and 1 ng/ml upregulated the number of TRACP-positive multinucleated cells after 7 days of co-culture of MLO-Y4 osteocytes and mouse bone marrow cells. Values are mean ± SEM from six separate experiments. CTL, control. ⁄Significant effect of IL-1b, p < 0.05.
3.2. IL-1b alters osteoclastogenesis-related gene expression by osteocytes Since IL-1b increased the formation of osteoclasts, we investigated whether IL-1b alters osteoclastogenesis-related gene expression by osteocytes. IL-1b (0.5 and 1 ng/ml) upregulated RANKL gene expression by 3.7- to 4.0-fold (Fig. 2A). However, IL-1b (0.5 and 1 ng/ml) downregulated OPG gene expression by 1.2- to 1.7fold (Fig. 2B). As a result IL-1b (0.5 and 1 ng/ml) upregulated RANKL/OPG ratio by 2.3- to 4.0-fold. IL-1b did not affect CYR61 gene expression (Fig. 2C). 3.3. Mechanical loading of osteocytes inhibits IL-1b stimulated osteoclastogenesis Next, we determined whether mechanical loading of osteocytes in the absence or presence of IL-1b affects osteoclast formation in a co-culture system of MLO-Y4 osteocytes and bone marrow cells.
To assess whether mechanical loading of osteocytes in the presence or absence of IL-1b affects CYR61, RANKL, and OPG gene expression, MLO-Y4 osteocytes were treated with or without 1 h PFF in the presence of IL-1b (0.1–1 ng/ml). PFF upregulated CYR61 gene expression by 1.3-fold in the absence of IL-1b (Fig. 4A). Addition of IL-1b (0.1–1 ng/ml) inhibited the upregulation of CYR61 gene expression after mechanical stimulation by PFF (Fig. 4A). PFF did not affect CYR61 gene expression in the presence of IL-1b (0.1–1 ng/ml) (Fig. 4A). PFF upregulated RANKL gene expression by 3.0-fold (Fig. 4B) and OPG gene expression by 1.4fold in the absence of IL-1b (Fig. 4C). Addition of IL-1b (0.5 and 1 ng/ml) inhibited the upregulation of OPG gene expression after mechanical stimulation by PFF (Fig. 4C). PFF upregulated RANKL gene expression by 1.6-fold in the presence of IL-1b (0.1 ng/ml), but did not affect OPG gene expression in the presence of IL-1b (0.1–1 ng/ml).
4. Discussion In the present study we found that IL-1b increases osteocytemodulated osteoclastogenesis. These results are in accordance with data by others showing that IL-1b indirectly facilitates osteoclastogenesis by acting on osteoblasts or human periodontal ligament fibroblasts [24,25]. We found that IL-1b upregulates RANKL and inhibits OPG gene expression by osteocytes. Our results show that IL-1b probably stimulates osteoclastogenesis by modulating RANKL and OPG gene expression by osteocytes. Alternatively the
Fig. 2. Effect of IL-1b on RANKL, OPG, and CYR61 gene expression by MLO-Y4 osteocytes. Cells were incubated with IL-1b (0.1–1 ng/ml) for 24 h. (A) IL-1 b at 0.5 and 1 ng/ml upregulated RANKL gene expression. (B) IL-1 b at 0.5 and 1 ng/ml downregulated OPG gene expression. (C) IL-1 b did not affect CYR61 gene expression. Values are mean ± SEM of IL-1b treated-over-control ratios from five different experiments. Dashed line, T/C = 1 (no effect of IL-1b). ⁄Significant effect of IL-1b, p < 0.05.
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Fig. 3. Effect of conditioned medium obtained from static or mechanically stimulated osteocytes treated with or without IL-1b on osteocyte-modulated osteoclastogenesis. Cells were treated with IL-1b for 24 h before application of PFF. (A) Conditioned medium from static osteocytes treated with 0.1, 0.5, or 1 ng/ml IL-1b significantly upregulated the number of TRACP-positive multinucleated cells, whereas conditioned medium from mechanically stimulated osteocytes treated with 0.1, 0.5, or 1 ng/ml IL-1b significantly inhibited the number of TRACP-positive multinucleated cells. (B) Conditioned medium from mechanically stimulated osteocytes inhibited the formation of osteoclasts. Values are mean ± SEM from four separate experiments. PFF, pulsating fluid flow; static, static control conditions; CTL, control. ⁄Significant effect of PFF, p < 0.05. # Significant effect of IL1-b, p < 0.05.
increased formation of osteoclast-like cells might have been due to osteocyte apoptosis [26]. Osteocytes extend their cell processes into bone marrow spaces [27], where osteoclast precursors are present. It is likely that osteocytes communicate with these osteoclast precursors present in the bone marrow space via their dendritic processes. Therefore we used a co-culture model of MLO-Y4 osteocytes and mouse bone marrow cells, which likely mimics the in vivo situation. Soluble factors released by mechanically loaded osteocytes inhibit osteoclastogenesis induced by stromal cells and osteocytes [28,29]. Therefore we investigated if conditioned medium from mechanically loaded and IL-1b-treated osteocytes does modulate osteoclastogenesis. We found that conditioned medium from static IL-1b-treated osteocytes dose-dependently increased the number of osteoclasts in co-cultures of osteocytes and bone marrow cells. Conversely conditioned medium from mechanically loaded IL-1b-treated osteocytes inhibited the formation of osteoclasts. To our knowledge we show for the first time that mechanical loading of osteocytes in the presence of IL-1b abolishes osteoclastogenesis. Since osteocytes are the most abundant bone cell type, vastly outnumbering the osteoblasts and osteoclasts in the bone, our findings provide important information related to the prevention of IL-1b-induced bone loss. Mechanically loaded osteocytes produce several factors which inhibit osteoclastogenesis. Osteocytes produce high levels of nitric oxide in response to mechanical stimulation [15,30], and nitric oxide mediates the inhibition of osteoclast activity by mechanically stimulated osteocytes [17]. Although IL-1b inhibits the upregulation of nitric oxide production after mechanical stimulation by PFF [18], we found that mechanical loading of osteocytes in the
presence of IL-1b inhibits osteoclastogenesis. We propose that the inhibition of IL-b-induced osteoclastogenesis by mechanically loaded osteocytes is independent of nitric oxide. Since a decrease in the RANKL/OPG ratio inhibits osteoclastogenesis, we also investigated whether mechanical loading of osteocytes in the presence of IL-1b modulates the RANKL/OPG ratio in osteocytes. We found that mechanical stimulation in the presence of IL-1b did not decrease the RANKL/OPG ratio in osteocytes. This suggests that mechanical loading of osteocytes in the presence of IL-1b might inhibit osteoclastogenesis in a RANKL/RANK/OPG independent manner. We then determined the effect of mechanical loading on CRY61 gene expression, a negative regulator of osteoclastogenesis [11], in osteocytes. Under these conditions we found an upregulation of CYR61 gene expression, which has not been shown before. Since CYR61 has been shown to inhibit osteoclastogenesis in a RANKL/RANK/OPG independent manner [11], this suggests that CYR61 might be a soluble factor produced after mechanical loading of osteocytes in the absence of IL-1b leading to the inhibition of osteoclastogenesis. However we found that IL-1b inhibits the upregulation of CYR61 gene expression after PFF. In spite of the inhibited expression of CYR61, conditioned medium from mechanically loaded IL-1b treated osteocytes still inhibited the formation of osteoclasts. Therefore CYR61 might not be involved in the inhibition of IL-1b-induced osteoclastogenesis. We also found that conditioned medium from mechanically loaded IL-1b-treated osteocytes inhibited clustering of osteoclast precursors (data not shown). This finding suggests the presence of factors in the conditioned medium that inhibit migration of osteoclast precursors towards each other to form osteoclasts.
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Fig. 4. Effect of 1 h PFF on CYR61, RANKL, and OPG gene expression by MLO-Y4 osteocytes in the presence or absence of IL-1b. (A) PFF upregulated CYR61 gene expression by osteocytes in the absence of IL-1b. (B) PFF upregulated RANKL gene expression by osteocytes in the absence and presence of 0.5 ng/ml IL-1b. (C) PFF upregulated OPG gene expression by osteocytes in the absence of IL-1b. Values are mean ± SEM from five different PFF experiments. PFF, pulsating fluid flow; static, static control conditions; CTL, control. ⁄Significant effect of PFF, p < 0.05. #Significant effect of IL1-b, p < 0.05.
Further research focusing on the identification of the specific soluble factors produced after mechanical loading of IL-1b-treated osteocytes that inhibit osteoclastogenesis is warranted. In conclusion, our results show that IL-1b increases osteocytemodulated osteoclastogenesis, whereas mechanical loading of osteocytes inhibits IL-1b-induced osteoclastogenesis. Based on these findings we propose that in patients with RA, limited movement or disuse as a result of pain and presence of IL-1b might enhance bone loss, whereas mechanical loading of bone in these patients will hopefully, at least in part, alleviate the negative effects of IL-1b on bone mass. Acknowledgments The work of R.N. Kulkarni was supported by a Grant from the University of Amsterdam. The Research Institute MOVE of the VU University Amsterdam supported the work of A.D. Bakker. The authors thank J.M.A. Hogervorst for excellent help in performing mechanical loading experiments. References [1] T. Nakashima, M. Hayashi, T. Fukunaga, et al., Evidence for osteocyte regulation of bone homeostasis through RANKL expression, Nat. Med. 17 (2011) 1231– 1234. [2] P.S. Vezeridis, C.M. Semeins, Q. Chen, J. Klein-Nulend, Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation, Biochem. Biophys. Res. Commun. 348 (2006) 1082–1088. [3] J. Xiong, M. Onal, R.L. Jilka, et al., Matrix-embedded cells control osteoclast formation, Nat. Med. 17 (2011) 1235–1241. [4] E.H. Burger, J. Klein-Nulend, Mechanotransduction in bone – role of the lacunocanalicular network, FASEB J. 13 (1999) S101–S112. [5] J.A. Eastgate, J.A. Symons, N.C. Wood, et al., Correlation of plasma interleukin 1 levels with disease activity in rheumatoid arthritis, Lancet 24 (1988) 706–709.
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